Mouse liver plasma membrane redox system activity is altered by aging and modulated by calorie restriction

Caloric restriction (CR) is known as the only non-genetic method proven to slow the rate of aging and extend lifespan in animals. Free radicals production emerges from normal metabolic activity and generates the accumulation of oxidized macromolecules, one of the main characteristics of aging. Due to its central role in cell bioenergetics, a great interest has been paid to CR-induced modifications in mitochondria, where CR has been suggested to decrease reactive oxygen species production. The plasma membrane contains a trans-membrane redox system (PMRS) that provides electrons to recycle lipophilic antioxidants, such as α-tocopherol and coenzyme Q (CoQ), and to modulate cytosolic redox homeostasis. In the present study, we have investigated age differences in the PMRS in mouse liver and their modulation by CR. Aging induced a decrease in the ratio of CoQ₁₀/CoQ₉ and α-tocopherol in liver PM from AL-fed mice that was attenuated by CR. CoQ-dependent NAD(P)H dehydrogenases highly increased in CR old mice liver PMs. On the other hand, the CoQ-independent NADH-FCN reductase activity increased in AL-fed animals; whereas, in mice under CR this activity did not change during aging. Our results suggest that liver PMRS activity changes during aging and that CR modulates these changes. By this mechanism CR maintains a higher antioxidant capacity in liver PM of old animals by increasing the activity of CoQ-dependent reductases. Also, the putative role of PMRS in the modulation of redox homeostasis of cytosol is implicated.     

Cytochrome b5 reductase,a plasma membrane redox enzyme,protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics

The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD⁺/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H₂O₂ (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H₂O₂ and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD⁺/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-015-9859-9) contains supplementary material, which is available to authorized users.     

Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver

Aging is associated with increased production of reactive oxygen species and oxidation-induced damage to intracellular structures and membranes. Caloric restriction (CR) is the only non-genetic method proven to extend lifespan in mammals. Although the mechanisms of CR remain to be clearly elucidated, reductions in oxidative stress have been shown to increase lifespan in several model systems. Oxidative stress can be attenuated by CR. Mitochondria and plasma membrane (PM) are normal sources of free radicals. The PM has a trans-membrane redox system that provides electrons to recycle lipophilic antioxidants, such as alpha-tocopherol and coenzyme Q (CoQ). The idea developed in this study is that the PM is intimately involved in cellular physiology controlling the relationship of the cell to its environment. PM is the key for protecting cellular integrity during aging. Specifically, we have investigated age-related alterations and the effects of CR in the trans-PM redox (antioxidant) system in rat liver. We found that age-related declines in the ratio of CoQ(10)/CoQ(9) and alpha-tocopherol in liver PM were attenuated by CR compared to those fed ad libitum (AL). CoQ-dependent NAD(P)H dehydrogenases were increased in CR old rat liver PMs. As a consequence, the liver PM of CR old rats was more resistant to oxidative stress-induced lipid peroxidation than AL rats. Thus, our results suggest that CR induces a higher capacity to oxidize NAD(P)H in the PM of old rat livers and as a result, a higher resistance to oxidative stress-induced damage.     

Effect of age and caloric restriction on coenzyme Q and alpha-tocopherol levels in the rat

Alterations in the amount of coenzyme Q and alpha-tocopherol during aging and in response to 40% reduction in caloric intake were determined in homogenates and mitochondria of liver, heart and kidney of the rat. A comparison among 4-, 19- and 28-month-old ad libitum fed (AL) rats indicated an age-related loss in the amount of CoQ9 and alpha-tocopherol in mitochondria of all the three tissues. Depletion of alpha-tocopherol, but not of CoQ, was also detectable in tissue homogenates, apparently due to the preferential sequestration of CoQ in the mitochondrial fraction. Comparison of 19-month-old AL and calorically restricted (CR) rats indicated that CR elevates the level of mitochondrial CoQ, but greatly diminishes the alpha-tocopherol content. Activity of DT-diaphorase, a quinone reductase, increased with age as well as in response to CR. Altogether, results are interpreted to suggest that the widely observed age-related increase in mitochondrial oxidative damage may be associated with depletion of CoQ and alpha-tocopherol, which are known to act in tandem to prevent oxidative damage to membranes.     

Coenzyme Q10 protects from aging-related oxidative stress and improves mitochondrial function in heart of rats fed a polyunsaturated fatty acid (PUFA)-rich diet

Coenzyme Q(10) supplementation on age-related changes in oxidative stress and function of heart mitochondria in rats fed a polyunsaturated fatty acid (PUFA)-rich diet was investigated. Two groups of rats were fed for 24 months on a PUFA-rich diet, differing in supplementation or not with coenzyme Q(10). Animals were killed at 6, 12, or 24 months. Fatty-acid profile, hydroperoxides, alpha-tocopherol, coenzyme Q, catalase and glutathione peroxidase activities, and cytochromes a+a(3), b, c+c(1) and cytochrome c oxidase activity were measured. Coenzyme Q(10)-supplemented animals showed lower hydroperoxide levels; higher content and/or activity of alpha-tocopherol, coenzyme Q, and catalase; and a slightly lower decrease in mitochondrial function. According to that, previously reported positive effects of coenzyme Q supplementation on the life span of rats fed a PUFA-rich diet might be a consequence, at least in part, of a lower oxidative stress level and perhaps, to a minor extent, of a smaller decrease in mitochondrial function.     

The plasma membrane redox system: a candidate source of aging-related oxidative stress

The plasma membrane redox system (PMRS) is an electron transport chain in the plasma membrane that transfers electrons from either intra- or extracellular donors to extracellular acceptors. Unlike the superoxide-generating NADPH oxidase of phagocytes and the homologous (but much less active) enzymes found in some other cells, the PMRS is still incompletely characterised at the molecular level. Much is known, however, concerning its function and affinity for both physiological and non-physiological substrates. A role for it in aging, the ‘reductive hotspot hypothesis’ (RHH), was proposed in 1998 as part of an explanation for the apparently indefinite survival in vivo of cells that have entirely lost mitochondrial respiratory capacity as a result of the accumulation of mitochondrial mutations. Stimulation of the PMRS might allow the cell to maintain redox homeostasis even while continuing to operate the Krebs cycle, which may be advantageous in many ways. However, the PMRS may, like the mitochondrial respiratory chain, be prone to generate superoxide when thus dysregulated – and in this case superoxide would be generated outside the cell, where antioxidant defences are more limited than inside the cell and where much highly oxidisable material is present. Cascades of peroxidation chain reactions initiated by this process may greatly amplify the oxidative stress on the organism that is caused by rare mitochondrially mutant cells. Since such cells increase in abundance with aging (though remaining rare), this is an economical hypothesis to explain the rise in oxidative stress seen in (and generally believed to contribute substantially to) mammalian aging. In an extension of previously published accounts of RHH, I propose here that the lysosomal toxicity of oxidised cholesterol derivatives (oxysterols) may contribute to the toxicity of mitochondrial mutations by affecting lysosomal function in many cell types in the same way as they have been proposed to do in arterial macrophages.     

A critical period in lifespan of male rats coincides with increased oxidative stress

The oxidative stress theory of aging has provided the best possible explanation for the processes which accompany aging and has received much support, however, in the last few years there have been questions regarding the validity of this theory. We have conducted experiments to determine an array of oxidative stress parameters in blood of male rats at various intervals (1, 4, 8, 12, 18 and 24 months) during their entire lifespan. Established protocols were used to measure plasma antioxidant capacity, erythrocyte plasma membrane redox system (PMRS), lipid and protein oxidation in erythrocytes and plasma, and erythrocyte glutathione (GSH). Our results on the total plasma antioxidant potential, PMRS in erythrocytes, protein and lipid peroxidation, and intracellular reduced GSH provide evidence that oxidative stress is minimal till approximately one-third of the total lifespan (8 months) and there is a spurt in oxidative stress between 8 and 12 months. The identification of a period (corresponding to 8–12 months) in the lifespan of rats coinciding with an spurt in oxidative stress is an interesting finding. No such report is available in humans or in any other model systems during aging.     

Erythrocyte plasma membrane redox system in human aging

Eukaryotic cells display a plasma membrane redox system (PMRS) that transfers electrons from intracellular substrates to extracellular electron acceptors. The physiologic importance of PMRS is still not fully understood. The authors have carried out studies to determine the activity of PMRS in human erythrocytes as a function of age and correlate the activity with total plasma antioxidant capacity in an effort to understand the role of PMRS in human aging. The study was carried out on 80 normal healthy subjects of both genders between the ages of 18 and 85 years. The activity of erythrocyte PMRS was estimated by following the reduction of ferricyanide. The total antioxidant capacity of the plasma was estimated in terms of Ferric Reducing Ability of Plasma (FRAP) values. A significant (p < 0.0001) positive correlation (r = 0.7797) is observed between PMRS activity of erythrocytes and human age. There is an age-dependent decrease in total plasma antioxidant capacity measured in terms of FRAP values. A highly significant correlation is observed between PMRS activity and plasma FRAP values. The authors hypothesize that the increased PMRS in erythrocytes during aging may be a protective mechanism of the system for efficient extracellular DHA reduction and ascorbate recycling under condition of increased oxidative stress.     

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.     

Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease

Alzheimer's disease (AD) is an age-related disorder characterized by deposition of amyloid beta-peptide (Abeta) and degeneration of neurons in brain regions such as the hippocampus, resulting in progressive cognitive dysfunction. The pathogenesis of AD is tightly linked to Abeta deposition and oxidative stress, but it remains unclear as to how these factors result in neuronal dysfunction and death. We report alterations in sphingolipid and cholesterol metabolism during normal brain aging and in the brains of AD patients that result in accumulation of long-chain ceramides and cholesterol. Membrane-associated oxidative stress occurs in association with the lipid alterations, and exposure of hippocampal neurons to Abeta induces membrane oxidative stress and the accumulation of ceramide species and cholesterol. Treatment of neurons with alpha-tocopherol or an inhibitor of sphingomyelin synthesis prevents accumulation of ceramides and cholesterol and protects them against death induced by Abeta. Our findings suggest a sequence of events in the pathogenesis of AD in which Abeta induces membrane-associated oxidative stress, resulting in perturbed ceramide and cholesterol metabolism which, in turn, triggers a neurodegenerative cascade that leads to clinical disease.     

Methionine restriction decreases endogenous oxidative molecular damage and increases mitochondrial biogenesis and uncoupling protein 4 in rat brain

Aging plays a central role in the occurrence of neurodegenerative diseases. Caloric restriction (CR) mitigates oxidative stress by decreasing the rate of generation of endogenous damage, a mechanism that can contribute to the slowing of the aging rate induced by this intervention. Various reports have recently linked methionine to aging, and methionine restriction (MetR) without energy restriction also increases life span. We have thus hypothesized that MetR can be responsible, at least in part, for the decrease in endogenous oxidative damage in CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their life span. We have found that MetR: (1) decreases the mitochondrial complex I content and activity, as well as complex III content, while the complex II and IV, the mitochondrial flavoprotein apoptosis-inducing factor (AIF) and ATP content are unchanged; (2) increases the mitochondrial biogenesis factor PGC-1alpha; (3) increases the resistance of brain to metabolic and oxidative stress by increasing mitochondrial uncoupling protein 4 uncoupling protein 4 (UCP4); and (4) decreases mitochondrial oxidative DNA damage and all five different markers of protein oxidation measured and lowers membrane unsaturation in rat brain. No changes were detected for protein amino acid composition. These beneficial MetR-induced changes likely derived from metabolic reprogramming at the cellular and tissue level can play a key role in the protection against aging-associated neurodegenerative disorders.     

Oxidative stress in the brain of reproductive male rats during aging

Reproduction alters the male physiology. We performed a comprehensive study to examine oxidative stress in the brains of male rats with (experienced) or without (naïve) reproductive activity during aging. Oxidative stress was assessed by measuring the activity of catalase, glutathione peroxidase, superoxide dismutase, glutathione S-transferase, aconitase, and aconitase reactivated, and by measuring lipid peroxidation, protein carbonylation, nitrite and nitrate levels, vitamin C levels, and glutathione (total, reduced, oxidized forms) levels in brain tissue, as well as testosterone and estradiol levels in serum. Reproductively active animals exhibited increased testosterone levels and aconitase activity, suggesting an increased metabolism. Increased antioxidant enzyme activities and increased levels of antioxidant compounds were observed, yet damage to biomolecules was also observed in experienced rats. During aging changes in oxidative stress were observed. We found higher activities of antioxidant enzymes, higher amounts of antioxidants, and more damage at six months of age among experienced animals than among naïve animals. Similar antioxidant activities and levels, and damage were found between the groups at twenty-four months of age. These results add comprehensive data regarding changes in oxidative stress during aging, and suggest an explanation for the costs of reproduction.     

Environmental contaminants and redox status of coenzyme Q10 and vitamin E in Inuit from Nunavik

The Inuit are heavily exposed to potentially prooxidant contaminants such as methylmercury (MeHg) and polychlorinated biphenyls (PCB) through their traditional diet. This diet is also an abundant source of n-3 polyunsaturated fatty acids (n-3 PUFA), selenium, and antioxidants, which might reduce cardiovascular risk. Although Inuit from Nunavik have low concentrations of plasma oxidized low-density lipoprotein (OxLDL) and elevated glutathione-related antioxidant defenses, the variance in OxLDL was predicted by PCB and blood glutathione, leaving the issue of contaminant-associated oxidative stress unresolved. The objective of the study was to assess oxidative stress in these Inuit by measuring the plasma concentrations and redox states of alpha-tocopherol and coenzyme Q10 (CoQ10), 2 sensitive biomarkers of oxidative stress, in relation to exposure. Plasma lipophilic antioxidants were determined by high-performance liquid chromatography-coupled electrochemical detection; and their relations to PCB, MeHg, n-3 PUFA, selenium, and OxLDL were assessed by multivariate analyses. Ubiquinol-10, ubiquinone-10, and ubiquinone-10 to CoQ10(total) ratio were elevated as compared with white populations but showed no associations with PCB, MeHg, or n-3 PUFA. Ubiquinol-10 (beta = .23, P = .007) and CoQ10(total) (beta = .27, P = .009) were predicted by blood selenium; and alpha-tocopherol, by PCB (beta = 4.12, P = .0002), n-3 PUFA (beta = 9.16, P = .02), and OxLDL (beta = 3.04, P = .05). Unexpectedly, the alpha-tocopheryl quinone to alpha-tocopherol ratio, in the reference range, was negatively predicted by PCB (beta = -0.41, P = .02). Using sensitive biomarkers of redox alterations, we found no evidence for MeHg- or PCB-associated oxidative stress in these Inuit. However, despite robust blood antioxidant defenses, the unusually elevated ubiquinone-10 to CoQ10(total) ratio (0.21 +/- 0.11) suggests some form of oxidative stress of unknown origin.     

Modifications of plasma proteome in long-lived rats fed on a coenzyme Q10-supplemented diet

Dietary coenzyme Q(10) prolongs life span of rats fed on a PUFAn-6-enriched diet. Our aim was to analyze changes in the levels of plasma proteins of rats fed on a PUFAn-6 plus coenzyme Q(10)-based diet. This approach could give novel insights into the mechanisms of life span extension by dietary coenzyme Q(10) in the rat. Serum albumin, which decreases with aging in the rat, was significantly increased by coenzyme Q(10) supplementation both at 6 and 24 months. After depletion of the most abundant proteins by affinity chromatography, levels of less abundant plasma proteins were also studied by using 2D-electrophoresis and MALDI-TOF mass fingerprinting analysis. Our results have shown that lifelong dietary supplementation with coenzyme Q(10) induced significant decreases of plasma hemopexin, apolipoprotein H and inter-alpha-inhibitor H4P heavy chain (at both 6 and 24 months), preprohaptoglobin, fibrinogen gamma-chain precursor, and fetuin-like protein (at 6 months), and alpha-1-antitrypsin precursor and type II peroxiredoxin (at 24 months). On the other hand, coenzyme Q(10) supplementation resulted in significant increases of serine protease inhibitor 3, vitamin D-binding protein (at 6 months), and Apo A-I (at 24 months). Our results support a beneficial role of dietary coenzyme Q(10) decreasing oxidative stress and cardiovascular risk, and modulating inflammation during aging.     

Antimalarial Quinones for Prophylaxis Based on a Rationale of Inhibition of Electron Transfer in Plasmodium

Knowledge of the biochemistry of Plasmodium is emerging as a new field. Previous studies showed that the parasite apparently requires electron transfer for energy, and techniques to study such energy mechanisms are available. The discovery of the existence of coenzyme Q(8) in Plasmodium implies an indispensable functionality for this redox entity in the electron transfer of the parasite, as coenzyme Q(n) similarily functions in other forms of life.Effective antimalarial activity in prophylaxis has been demonstrated in sporozoite-induced infections by Plasmodium gallinaceum in chicks by several representatives of 7-alkylmercapto-6-hydroxy-5,8-quinolinequinones. The absence of toxicity in this assay even at greatly elevated dosage underscores the achievement of selectivity and safety to the host for the potential utilization of antimetabolites of coenzyme Q(n) as medicinals.Seven new 7-alkylmercapto-6-hydroxy-5,8-quinoline-quinones were synthesized. The structural variations of the 7-alkylmercapto group in relationship to the antimalarial activities reveal substantial differences in biological activities, which can reflect molecular specificities of enzyme sites and which are not evident from the deceptively minor structural differences in the alkylmercapto groups.These analogs of coenzyme Q(8) having effective antimalarial activity are known to inhibit mammalian coenzyme Q(n) enzymes, and could be useful in elucidation of the basic electron transfer mechanisms of Plasmodium.     

Effect of coenzyme q10 on myopathic symptoms in patients treated with statins

Treatment of hypercholesterolemia with statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) is effective in the primary and secondary prevention of cardiovascular disease. However, statin use is often associated with a variety of muscle-related symptoms or myopathies. Myopathy may be related in part to statin inhibition of the endogenous synthesis of coenzyme Q10, an essential cofactor for mitochondrial energy production. The aim of this study is to determine whether coenzyme Q10 supplementation would reduce the degree of muscle pain associated with statin treatment. Patients with myopathic symptoms were randomly assigned in a double-blinded protocol to treatment with coenzyme Q10 (100 mg/day, n = 18) or vitamin E (400 IU/day, n = 14) for 30 days. Muscle pain and pain interference with daily activities were assessed before and after treatment. After a 30-day intervention, pain severity decreased by 40% (p <0.001) and pain interference with daily activities decreased by 38% (p <0.02) in the group treated with coenzyme Q10. In contrast, no changes in pain severity (+9%, p = NS) or pain interference with daily activities (-11%, p = NS) was observed in the group treated with vitamin E. In conclusion, results suggest that coenzyme Q10 supplementation may decrease muscle pain associated with statin treatment. Thus, coenzyme Q10 supplementation may offer an alternative to stopping treatment with these vital drugs.     

Effects of caloric restriction on post-spawning death of ayu

Caloric restriction (CR) is the only established intervention that extends life span in mammals, insects and nematodes. One of the hypotheses suggested that most of the effects of CR on aging may be due to reduced oxidative stress at the cellular level. It was known that ayu (Plecoglossus altivelis) produced ROS higher than other fish and that the life span of ayu is only one year. The present study attempts to quantify age-associated changes of the degree of attenuation on oxidative damage and hormonal homeostases in CR. The levels of 8-OHdG as the oxidative DNA damage level and the caspase-9/6, -3-like activities as the induction factors of apoptosis with aging in brain and liver were surveyed. Caspase-like activities in brain and liver were reduced by CR, while CR had no influence on DNA damage level. However, life span of ayu was not prolonged by CR. These results suggested that there would be factors determining life span of ayu other than CR and apoptosis.